DESCRIPTION: New Photostable Nanoprobes for Real-time Imaging of Single Live Cells. Different types of cells express trace amounts of distinctive sets of receptors, enabling them serve as biomarkers for cell identification. Binding of a few ligand molecules with its receptors on single live cells can initiate dynamic cascades of cellular signaling pathways, alter cellular functions and cause diseases. Multiple types of ligand-receptor (L-R) interactions typically work together to regulate and alter the cellular functions. Such signaling pathways can take minutes, hours or days. Thus, it is crucial to simultaneously visualize different types of receptors on single live cells with single-molecule sensitivity in order to quantify various types of receptor molecules, to directly capture how L-R interactions work together to achieve given cellular functions, and to effectively detect rare subsets of single live cells. Currently, fluorescence microscopy using fluorescence imaging probes is the primary workhorse for live cell imaging. Unfortunately, fluorescence probes (fluorophor, fluorescence protein, QD) suffer intrinsic photobleaching, making them unable to continuously capture the dynamic events of single live cells over hours. Photobleaching also makes quantitative analysis over time difficult. Separation of different excitation and emission of various fluorophores leads to complex and expensive instrument and restricts their capacity of multiplexing study of multiple types of molecules simultaneously. All fluorescence imaging methods including conventional, confocal and super-resolution fluorescence microscopy suffer these same fundamental limitations. In this proposal, we aim to develop a novel molecular imaging platform including photostable multicolored and multifunctional single molecule nanoparticle optical biosensors (SMNOBS) and far-field photostable optical nanoscopy (PHOTON) for real-time study of functions of single live cells. To demonstrate the-proof-of-concept, we will use them to quantitatively image and molecular characterize roles and functions of multiple types of the receptors on rare subsets of ovarian cancer stem cells (oCSCs) in highly heterogeneous tumor cell populations and to study their differentiation mechanisms. These powerful new tools are expected to address a wide range of pressing biochemical and biomedical questions about molecular and real-time characterization of functions of single receptor molecules on single live cells and their related signaling pathways in real time. The proposed study will enable us to create an interdisciplinary educational environment for students; expose them to leading-edge biomedical research program; involve more students in biomedical research activities; enhance and strengthen institutional biomedical research program and infrastructure.